Super easy and cheap method for making electrocompetent Gram -ve bacteria!

[Charles Vander Broek]

Hi All,

I just discovered DIY biology today and I am really excited for it's implications!  I'm a microbiologist and I tend to try to find the fastest and easiest (read lazy) ways to work in the lab.  I figure I will start by sharing my favorite method for making electrocompetent gram -ve bacteria.  This method has worked very well for me (sometimes better than commercial cells) using E. coli, Pseudomonas spp., and Burkholderia spp.  This method takes about 10-15 min, requires NO CHILLING, NO OD READINGS, and NO EXPENSIVE CHEMICALS which always pissed me off.  

Reagents:

O/N culture of bacteria (Can be stationary or exponential phase, doesn't matter.  About 6 ml of stationary phase E. coli is enough for 2x 50 ul transformations).

DI water

Sucrose (Now this is where it gets interesting. I have always used laboratory grade D-sucrose, but I think table sugar should do just fine as it is around 99.8-99.9% sucrose and should have low enough ion concentrations that it should work.  If someone tries this let me know how it works and I will give it a try as well!)

Equipment:

1.5ml microfuge tubes

Table top Microfuge

Optional: A larger cetrifuge and tubes for bulking up the procedure for large quantities of transformations.

Procedure:

1. Prepare 300mM sucrose in the DI Water

2. Spin down the bacteria.  6ul can be split into 4x 1.5 ml microfuge tubes or spun in a larger tube.  The spin should be enough to pellet the bacteria for removal of the supernatant (ie      13,000 RPM for 2 min in a tabletop microfuge)

3. Remove the supernatant and re-suspend the bacteria in 1 ml of 300 mM sucrose. The bacteria split in the 4 tubes can be combined at this point (suspend all 4 pellets in the same 1ml of  200 mM sucrose).

4. Pellet the bacteria at 13,000 RPM for 2 min in a table to microfuge.

5. Remove the supernatant and re-suspend the pellet well in 100 ul of 300 mM sucrose.

6. The bacteria are now ready to be electroporated.  I usually split the pellet in two 50 ul alliquots and add about 100-200 ng of plasmid DNA to the 50 ul of bacteria.

7. I transform using a 1 cm cuvette at 1.8 kV.

If you are getting sparks try adding one extra wash in sucrose.  Also I suspend my DNA in DI water so if you are suspending in a solution with ions this may affect your electroporations.

Good luck and happy trails!  If you need any more info let me know!

The paper that this detailed in!  These guys made my life so much easier :):

J Microbiol Methods. 2006 Mar;64(3):391-7. Epub 2005 Jun 28.

A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation.

Choi KH, Kumar A, Schweizer HP.

Source

Department of Microbiology, Immunology and Pathology, Colorado State University, 1682 Campus Delivery, Fort Collins, CO 80523, USA.

Abstract

A rapid microcentrifuge-based method is described for preparation of Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold increased transformation efficiencies over existing procedures. This increased efficiency now enables the use of transformation for all applications requiring DNA transfer. These include transfer of chromosomal mutations marked with antibiotic resistance genes between P. aeruginosa strains, which solves the riddle of not having an efficient and reliable transduction procedure for this bacterium. Not surprisingly, the method also allows for very efficient transformation with replicative plasmids, with transformation efficiencies ranging from 10(7) to >10(11) transformants per microgram of DNA. Lastly, with efficiencies of up to >10(3) transformants per microgram of DNA the method replaces in most instances conjugation for the transfer of non-replicative plasmids used in gene replacement, site-specific gene integration and transposon mutagenesis experiments.

PMID:

 

15987659

 

[PubMed - indexed for MEDLINE]

[Mac Cowell]

Awesome protocol, thanks for sharing.

What kind of electroporator do you use? They are expensive...

How do you estimate DNA concentrations - 280/260 ratio? If so, what kind of spec do you use?

Patrick d'Haeseleer and others have had some success sonoporating cells with low-cost jewelry sonicators. Not sure what the prep is first. I think it's easy:

https://groups.google.com/d/msg/diybio/1hklOcZgT20/WGPjM3vH07QJ

You should try it! Maybe Patrick will chime in with more details.

Mac

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[Charles Vander Broek]

This is proof that I am new to this!  I should have known ALL lab equipment is very expensive.  I have used 3 different electroporators ranging from new to very old with this protocol with similar results.  I will take a look at the specifics at work tomorrow.  A lot of my work is done in a high containment laboratory so we often have to alter protocols to not involve cooling or running water/water baths. 

As far as estimating DNA concentration I usually do it by running a small amount of DNA on an agarose gel alongside a known amount of control.  This has the advantage of showing me that my DNA is still in good condition.  I also sometimes use 260/280nm witha  nanodrop spec.

I would love to give the sonication method a go.  I will have to see if I can find a sonicator.  

This is all just too cool.  I have a really nice method for protein precipitation I will put up as well.  Also I think bacterial expression of single chain antibodies may prove useful for a cheap alternative that doesn't require animals.  The problem is I cant think of an easy way to purify it.

[Nathan McCorkle]

I've simply rinsed the overnight e.coli culture a few times with
sterile distilled water then electroporated, and proved better
transformation efficiency than using CaCl and heat shock.

Just started with the eppendorf full of overnight culture,
centrifuged, decanted and resuspended in DI H2O... repeated the pellet
rinsing twice more, added normal amount of plasmid DNA, electroporated
with GenePulser in a 0.1cm cuvette at 1.8kV.

See here for more details:
https://groups.google.com/d/msg/diybio/JcKyNIQGLIM/u4qIOllQRnAJ

On Thu, Oct 17, 2013 at 10:26 AM, Charles Vander Broek
<charles.v...@gmail.com> wrote:

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[Charles Vander Broek]

The only worry would be exposing the cells to such a hypotonic environment at room temperature would cause quite a lot of cell lysis.  That being said, with an easy to use strain and enough cells loosing a few doesn't matter, especially if you are getting such good efficiency!  Thanks for the info, will give that a try with a few strains myself!  I have been meaning to try the sucrose method with a few Gram +ve cells, so I may try the DI Water as well!

[Cathal Garvey (Phone)]

Heh, when enough cells lyse, they make the solution isotonic to the rest! :) I imagine excessive lysis ups the salt contents and likelihood of electrode arcing though? Never did much EP myself.

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